The present invention concerns a method of using the BK enhancer in the presence of an immediate-early gene product of a large DNA virus in increase transcription of a recombinant gene in eukaryotic host cells. The BK enhancer is a defined segment of DNA that consists of three repeated sequences (the prototye BK enhancer is depicted in Example 17, below). However, a wide variety of BK enhancer variants, not all consisting of three repeated sequences, are known in the art and suitable for use in the invention.
The BK enhancer sequence exemplified herein is obtained from BK virus, a human papovavirus that was first isolated from the urine of an immunosuppressed patient. BK virus is suspected of causing an unapparent childhood infection and is ubiquitous in the human population. Although BK virus grows optimally in human cells, the virus undergoes an abortive cycle in non-primate cells, transforms rodent cells in vitro, and induces tumors in hamsters. BK virus is very similar to SV40, but the enhancer sequences of the two papovaviruses, SV40 and BK, differ substantially in nucleotide sequence. The complete nucleotide sequence of BK virus (xcx9c5.2 kb) has been disclosed by Seif et al., 1979, Cell 18:963, and Yang and Wu, 1979, Science 206:456. Prototype BK virus is available from the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Md. 20852-1776, under the accession number ATCC VR-837. A restriction site and function map of prototype BK virus is presented in FIG. 1 of the accompanying drawings.
Enhancer elements are cis-acting and increase the level of transcription of an adjacent gene from its promoter in a fashion that is relatively independent of the position and orientation of the enhancer element. In fact, Khoury and Gruss, 1983, Cell 33:313, state that xe2x80x9cthe remarkable ability of enhancer sequences to function upstream from, within, or downstream from eukaryotic genes distinguishes them from classical promoter elements . . . xe2x80x9d and suggest that certain experimental results indicate that xe2x80x9cenhancers can act over considerable distances (perhaps  greater than 10 kb).xe2x80x9d
The present invention teaches that unexpected increases in transcription result upon positioning the BK enhancer immediately upstream of (on the 5xe2x80x2 side of) the xe2x80x9cCAATxe2x80x9d region of a eukaryotic promoter that is used in tandem with the BK enhancer to transcribe a DNA sequence encoding a useful substance. The CAAT region or xe2x80x9cimmediate upstream regionxe2x80x9d or xe2x80x9c-80 homology sequencexe2x80x9d is a cis-acting upstream element that is a conserved region of nucleotides observed in promoters whose sequences for transcriptional activity have been dissected. The CAAT region is found in many, but not all, promoters. In other promoters, equivalent cis-acting upstream elements are found, including SP1 binding sites, the octa sequence, nuclear factor 1 binding sits, the AP1 and AP2 homologies, glucocorticoid response elements, and heat shock response elements. The CAAT region equivalent in the adenovirus major late promoter is the upstream transcription factor (UTF) binding site (approximate nucleotides xe2x88x9250 to xe2x88x9265 upstream of the CAP site). The CAAT sequence mediates the efficiency of transcription and, with few exceptions, cannot be deleted without decreasing promoter strength.
Enhancer elements have been identified in a number of viruses, including polyoma virus, papiloma virus, adenovirus, retrovirus, hepatitis virus, cytomegalovirus, herpes virus, papovaviruses, such as simian virus 40 (SV40) and BK, and in many non-viral genes, such as within mouse immunoglobulin gene introns. Enhancer elements may also be present in a wide variety of other organisms. Host cells often react differently to different enhancer elements. This cellular specificity indicates that host gene products interact with the enhancer element during gene expression.
Enhancer elements can also interact with viral gene products present in the host cell. Velcich and Ziff, 1983, Cell 40:705; Borrelli et al., 1984, Nature 312:608; and Hen et al., 1985, Science 230:1391, disclose that the adenovirus-2 early region 1A (E1A) gene products repress activation of transcription induced by the SV40, polyoma virus, mouse immunoglobulin gene and adenovirus-2 E1A enhancers. Eukaryotic expression vectors that utilized enhancers to increase transcription of recombinant genes consequently were not expected to work better than vectors without enhancers in E1A-containing host cells. In striking contrast to the prior art methods of using enhancers, the present method for using the BK virus enhancer element involves using the E1A gene product or a similar immediate-early gene product of a large DNA virus to maximize gene expression. Thus, the present invention teaches that the ability of the BK enhancer to promoter transcription of DNA is increased in the presence of the E1A gene product of any adenovirus.
The E1A gene product (actually, the E1A gene produces two products, which are collectively referred to herein as xe2x80x9cthe E1A gene productxe2x80x9d) is an immediate-early gene product of adenovirus, a large DNA virus. The present invention encompasses the use of any immediate-early gene product of a large DNA virus that functions similarly to the E1A gene product to increase the activity of the BK enhancer. The herpes simplex virus ICP4 protein, described by DeLuca et al., 1985, Mol. Cell. Biol. 5: 1997-2008, the pseudorabies virus IE protein, described by Feldman et al., 1982 P.N.A.S. 79:4952-4956, and the E1B protein of adenovirus are all immediate-early gene products of large DNA viruses that have functions similar to the E1A protein. Therefore, the method of the present invention includes the use of the ICP4, IE, or E1B proteins, either in the presence or absence of E1A protein, to increase the activity of the BK enhancer.
The present invention concerns a method of using the BK virus enhancer in the presence of an immediate-early gene product of a large DNA virus, such as the EIA gene product of adenovirus, for purposes of increasing transcription and expression of recombinant genes in eukaryotic host cells. Another significant aspect of the present invention relates to a variety of expression vectors that utilize the BK enhancer sequence in tandem with a eukaryotic promoter, such as the adenovirus late promoter (MLP), to drive expression of useful products in eukaryotic host cells. Many of these expression vectors comprise a BK enhancer-adenovirus late promoter cassette, which can be readily transferred to other vectors for use in the present method. The versatility of the present expression vectors is demonstrated by the high-level expression driven by these vectors of such diverse proteins as chloramphenicol acetyltransferase, protein C, tissue plasminogen activator, and modified tissue plasminogen activator.
In the construction of certain vectors of the invention, the BK enhancer and SV40 enhancer were placed in tandem at the front (5xe2x80x2) end of the MLP, itself positioned to drive expression of a recombinant gene on a recombinant DNA expression vector. This tandem placement yielded unexpectedly higher levels of expression in cells that did not express the immediate-early gene product of a large DNA virus. Consequently, a further aspect of the invention is a method of producing a gene product in a recombinant host cell that comprises transforming the host cell with a recombinant DNA vector that comprises two different enhancers placed at the 5xe2x80x2 end of the coding sequence for the gene product and culturing the transformed cell under conditions that allow for gene expression.
The practice of the invention to express human protein C in adenovirus-transformed cells led to the discovery that such cells are especially preferred hosts for the production of xcex3-carboxylated proteins. Consequently, a further aspect of the invention comprises a method for making xcex3-carboxylated proteins.
Yet another important aspect of the present invention concerns a method of increasing the activity of the BK enhancer relative to an adjacent eukaryotic promoter and is illustrated using the BK enhancer-adenovirus-2 late promoter cassette. These derivatives were constructed by enzymatic treatment that positioned the BK enhancer very close to the CAAT region of the adenovirus-2 late promoter. Dramatic increases in expression levels, as compared with constructions that lack this positioning, were observed when these modified BK enhancer-adenovirus late promoter sequences were incorporated into expression vectors and then used to drive expression of useful gene products in eukaryotic host cells. Thus, the present invention provides a method for increasing the activity of the BK enhancer relative to an adjacent eukaryotic promoter that comprises positioning the enhancer immediately upstream, within 0 to about 300 nucleotides, of the 5xe2x80x2 end of the CAAT region or CAAT region equivalent of the eukaryotic promoter.
Yet another aspect of the invention results from attempts to increase expression of recombinant products encoded on the vectors described herein by incorporation of portions of the tripartite leader sequence of adenovirus into those expression vectors. Significant increases in expression result when the first part of the tripartite leader of adenovirus is encoded into a recombinant DNA expression vector, and such expression can be further increased in some situations by action of the VA gene product of adenovirus.
An additional aspect of the present invention concerns a method of amplification of genes in primate cells. The most widely used method for gene amplification employs the murine dihydrofolate reductase gene for selection and amplification in a dhfr deficient cell line. Human polypeptides often require post-translational modifications which occur most efficiently in primate cells, yet most primate cells cannot be directly selected or amplified using only the dhfr system. The present invention provides a method wherein the primate cells are first isolated using a directly selectable marker, then amplified using the dhfr system, thereby significantly increasing the expression levels from primate cells.
Another aspect of the present invention concerns novel recombinantly produced human protein C molecules which contain glycosylation patterns totally unlike the human protein C molecules derived from plasma. The novel recombinantly produced protein C molecules display functional activities which are quite different than plasma-derived human protein C. Furthermore, the recombinant human protein C molecules derived from 293 cells contain fewer sialic acid residues than the plasma-derived human protein C.
For purposes of the present invention, the following terms are as defined below.
Antibioticxe2x80x94a substance produced by a microorganism that, either naturally or with limited chemical modification, will inhibit the growth of or kill another microorganism or eukaryotic cell.
Antibiotic Resistance-Conferring Genexe2x80x94a DNA segment that encodes an activity that confers resistance to an antibiotic.
ApRxe2x80x94the ampicillin-resistant phenotype or gene conferring same.
Cloningxe2x80x94the process of incorporating a segment of DNA into a recombinant DNA cloning vector.
CmRxe2x80x94the chloramphenicol-resistant phenotype or gene conferring same.
dhfrxe2x80x94dihydrofolate reductase.
epxe2x80x94a DNA segment comprising the SV40 early promoter of the T-antigen gene, the T-antigen binding sites, and the SV40 origin of replication.
Eukaryotic promoterxe2x80x94any DNA sequence that functions as a promoter in eukaryotic cells.
HmRxe2x80x94the hygromycin-resistant phenotype or gene conferring same.
IVSxe2x80x94DNA encoding an intron, also called an intervening sequence.
Large DNA virusxe2x80x94a virus that infects eukaryotic cells and has a genome greater than xcx9c10 kb in size, i.e., any of the pox viruses, adenoviruses, and herpes viruses.
MLPxe2x80x94the major late promoter of adenovirus, which is also referred to herein as the late promoter of adenovirus.
NeoRxe2x80x94the neomycin resistance-conferring gene, which can also be used to confer G418 resistance in eukaryotic host cells.
orixe2x80x94a plasmid origin of replication.
pAxe2x80x94a DNA sequence encoding a polyadenylation signal.
Promoterxe2x80x94a DNA sequence that directs transcription of DNA into RNA.
Recombinant DNA Cloning Vectorxe2x80x94any autonomously replicating or integrating agent that comprises a DNA molecule to which one or more additional DNA segments can be or have been added.
Recombinant DNA Expression Vectorxe2x80x94any recombinant DNA cloning vector comprising a promoter and associated insertion site, into which a DNA molecule that encodes a useful product can be inserted and expressed.
Recombinant DNA Vectorxe2x80x94any recombinant DNA cloning or expression vector.
Repliconxe2x80x94any DNA sequence that controls the replication of a recombinant DNA vector.
Restriction Fragmentxe2x80x94any linear DNA generated by the action of one or more restriction enzymes.
rRNAxe2x80x94ribosomal ribonucleic acid.
Sensitive Host Cellxe2x80x94a host cell that cannot grow in the presence of a given antibiotic or other toxic compound without a DNA segment that confers resistance thereto.
Structural Genexe2x80x94any DNA sequence that encodes a polypeptide, inclusive of that DNA encoding the start and stop codons.
Structural Polypeptidexe2x80x94any useful polypeptide, including, but not limited to, human protein C, tissue plasminogen activator, insulin, thrombomodulin, factor Va or factor VIIIa.
TcRxe2x80x94the tetracycline-resistant phenotype or gene conferring same.
Transformantxe2x80x94a recipient host cell that has undergone transformation.
Transformationxe2x80x94the introduction of DNA into a recipient host cell.
tRNAxe2x80x94transfer ribonucleic acid.